Decomposition of protein experimental compressibility into intrinsic and hydration shell contributions

The experimental determination of protein compressibility reflects both the protein intrinsic compressibility and the difference between the compressibility of water in the protein hydration shell and bulk water. We use molecular dynamics simulations to explore the dependence of the isothermal compressibility of the hydration shell surrounding globular proteins on differential contributions from charged, polar, and apolar protein-water interfaces. The compressibility of water in the protein hydration shell is accounted for by a linear combination of contributions from charged, polar, and apolar solvent-accessible surfaces. The results provide a formula for the deconvolution of experimental data into intrinsic and hydration contributions when a protein of known structure is investigated. The physical basis for the model is the variation in water density shown by the surface-specific radial distribution functions of water molecules around globular proteins. The compressibility of water hydrating charged atoms is lower than bulk water compressibility, the compressibility of water hydrating apolar atoms is somewhat larger than bulk water compressibility, and the compressibility of water around polar atoms is about the same as the compressibility of bulk water. We also assess whether hydration water compressibility determined from small compound data can be used to estimate the compressibility of hydration water surrounding proteins. The results, based on an analysis from four dipeptide solutions, indicate that small compound data cannot be used directly to estimate the compressibility of hydration water surrounding proteins.     

Large-scale networks of hydration water molecules around proteins investigated by cryogenic X-ray crystallography.

The structures at protein-water interface, i.e. the hydration structure of proteins, have been investigated by cryogenic X-ray crystal structure analyses. Hydration structures appeared far clearer at cryogenic temperature than at ambient temperature, presumably because the motions of hydration water molecules were quenched by cooling. Based on the structural models obtained, the hydration structures were systematically analyzed with respect to the amount of water molecules, the interaction modes between water molecules and proteins, the local and the global distribution of them on the surface of proteins. The standard tetrahedral interaction geometry of water in bulk retained at the interface and enabled the three-dimensional chain connection of hydrogen bonds between hydration water molecules and polar protein atoms. Large-scale networks of hydrogen bonds covering the entire surface of proteins were quite flexible to accommodate to the large-scale conformational changes of proteins and seemed to have great influences on the dynamics and function of proteins. The present observation may provide a new concept for discussing the dynamics of proteins in aqueous solution.     

Structural details of urea binding to barnase: a molecular dynamics analysis.

BACKGROUND: The molecular mechanism of urea-induced protein unfolding has not been established. It is generally thought that denaturation results from the stabilizing interactions of urea with portions of the protein that are buried in the native state and become exposed upon unfolding of the protein. RESULTS: We have performed molecular dynamics simulations of barnase (a 110 amino acid RNase from Bacillus amyloliquefaciens) with explicit water and urea molecules at 300 K and 360 K. The native conformation was unaffected in the 300 K simulations at neutral and low pH. Two of the three runs at 360 K and low pH showed some denaturation, with partial unfolding of the hydrophobic core 2. The first solvation shell has a much higher density of urea molecules (water/urea ratio ranging from 2.07 to 2.73) than the bulk (water/urea ratio of 4.56). About one half of the first-shell urea molecules are involved in hydrogen bonds with polar or charged groups on the barnase surface, and between 15% and 18% of the first-shell urea molecules participate in multiple hydrogen bonds with barnase. The more stably bound urea molecules tend to be in crevices or pockets on the barnase surface. CONCLUSIONS: The simulation results indicate that an aqueous urea solution solvates the surface of a polypeptide chain more favorably than pure water. Urea molecules interact more favorably with nonpolar groups of the protein than water does, and the presence of urea improves the interactions of water molecules with the hydrophilic groups of the protein. The results suggest that urea denaturation involves effects on both nonpolar and polar groups of proteins.     

Stopped-flow study of anesthetic effect on water-transport kinetics through phospholipid membranes. Interfacial versus lipid core ligands

We have compared ligand effects between polar and apolar anesthetic molecules upon water transport across phospholipid membranes by kinetic analysis of the osmotic swelling rate, using a stopped-flow technique. Chloroform and 1-hexanol were used as interfacial ligands, and carbon tetrachloride and n-hexane were used as their counterparts, representing lipid core action. Because anesthetics transform the solid-gel membrane into a liquid-crystalline state, and because phospholipid membranes display an anomaly in permeability at the phase transition, dimyristoylphosphatidylcholine vesicles were studied at temperatures above the main phase transition to avoid this anomaly. All these molecules increased the osmotic swelling rate. However, a significant difference was observed in the activation energy, delta Ep, between polar and apolar molecules; delta Ep was almost unaltered by the addition of polar molecules (chloroform and 1-hexanol), whereas it was decreased by apolar molecules (carbon tetrachloride and n-hexane). The obtained results were analyzed in terms of the dissolution-diffusion mechanism for water permeation across the lipid membrane. It is suggested that polar molecules affect water permeability by altering the partition of water between the membrane interior and water phase, and apolar molecules affect it by altering both the partition and the diffusion of water within the membrane interior.     

The mechanism of the type III antifreeze protein action: a computational study

The random network model of water quantitatively describes the different hydration heat capacities of polar and apolar solutes in terms of differential distortions of the water-water hydrogen bonding angle in the first hydration shell. This method of hydration analysis is applied here to study the hydration of the wild type III thermal hysteresis protein from eel pout and three mutations at residue 16. Wild type and one mutant have full activity, the other two mutants have little or no anti-freeze (thermal hysteresis) activity. The analysis reveals significant differences in the hydration structure of the ice-binding site (centered on residue 16) among four proteins. For the A16T and A16Y mutants with reduced activity, polar groups have a typical polar-like hydration. For the wild type and mutant A16C with 100% of the wild type activity, polar groups have unusual, very apolar-like hydration. In the latter case, hydrating water molecules form a more ice-like pattern of hydrogen bonding on the ice-binding face, while in the former case water-water H-bonds are more distorted and more heterogenous. Overall, the binding surface of active protein strongly enhances the water tetrahedral structure, i.e. promotes ice-like hydration. It is concluded that the specific shape, residue size and clustering of both polar/apolar groups are essential for the binding surface to recognize, and preferentially interact with nascent ice crystals forming in liquid water.     

Water-protein interactions from high-resolution protein crystallography

To understand the role of water in life at molecular and atomic levels, structures and interactions at the protein-water interface have been investigated by cryogenic X-ray crystallography. The method enabled a much clearer visualization of definite hydration sites on the protein surface than at ambient temperature. Using the structural models of proteins, including several hydration water molecules, the characteristics in hydration structures were systematically analysed for the amount, the interaction geometries between water molecules and proteins, and the local and global distribution of water molecules on the surface of proteins. The tetrahedral hydrogen-bond geometry of water molecules in bulk solvent was retained at the interface and enabled the extension of a three-dimensional chain connection of a hydrogen-bond network among hydration water molecules and polar protein atoms over the entire surface of proteins. Networks of hydrogen bonds were quite flexible to accommodate and/or to regulate the conformational changes of proteins such as domain motions. The present experimental results may have profound implications in the understanding of the physico-chemical principles governing the dynamics of proteins in an aqueous environment and a discussion of why water is essential to life at a molecular level.     

Thermodynamics of amide hydrogen bond formation in polar and apolar solvents

We present the initial findings of a theoretical study of hydrogen bond formation between two formamide molecules in water and in carbon tetrachloride. These systems were chosen as the simplest models for secondary structure formation in the polar environment near the protein surface and the apolar environment of the protein interior. We have employed thermodynamic simulation methods to obtain absolute binding free energies and free energy profiles for the formation of peptide hydrogen bonds in the two solvents. We find that the amide hydrogen bond is stable by 8.4 kcal/mol in CCl4, and by 0.3 kcal/mol in water. Our results indicate also that the hydrogen-bonded dimer is 2.2 kcal/mol more stable in water than it is in CCl4. We compare our results with those from experiment, and discuss their use in interpreting mechanisms of protein folding.     

Computer simulation of hydration networks around amino acids

The networks of solvent hydrogen bonds around polar and apolar amino acids have been studied by computer simulation techniques using a non-pair additive model for the water molecules interactions. Analysis of the simulated aqueous solutions has shown the presence of water molecules which (a) form a bridge around individual polar solute atoms (self-bridging loops) and (b) form chains between different polar solute atoms (polar bridging chains). Some of these networks associated with polar solute atoms from pentagons but 4, 6 and 7 sided polygons are also seen. The water molecule close to apolar solute atoms (<4.0 Å) also form irregular networks with polygons of 4, 5, 6 and 7 sides. These networks are compared with those found experimentally in ice, clathrates and crystal hydrates of macromolecules.     

Hydrophobic tendency of polar group hydration as a major force in type I antifreeze protein recognition

The random network model of water quantitatively describes the different hydration heat capacities of polar and apolar solutes in terms of distortions of the water-water hydrogen bonding angle in the first hydration shell (Gallagher and Sharp, JACS 2003;125:9853). The distribution of this angle in pure water is bimodal, with a low-angle population and high-angle population. Polar solutes increase the high-angle population while apolar solutes increase the low-angle population. The ratio of the two populations quantifies the hydrophobicity of the solute and provides a sensitive measure of water structural distortions. This method of analysis is applied to study hydration of type I thermal hysteresis protein (THP) from winter flounder and three quadruple mutants of four threonine residues at positions 2, 13, 24, and 35. Wild-type and two mutants (VVVV and AAAA) have antifreeze (thermal hysteresis) activity, while the other mutant (SSSS) has no activity. The analysis reveals significant differences in the hydration structure of the ice-binding site. For the SSSS mutant, polar groups have a typical polar-like hydration, that is, more high-angle H-bonds than bulk water. For the wild-type and active mutants, polar groups have unusual, very apolar-like hydration, that is, more low-angle H-bonds than bulk water. This pattern of hydration was seen previously in the structurally distinct type III THPs (Yang & Sharp Biophys Chem 2004;109:137), suggesting for the first time a general mechanism for different THP classes. The specific shape, residue size, and clustering of both polar and apoler groups are essential for an active ice binding surface.     

Protein-water interactions in ribonuclease A and angiogenin: a molecular dynamics study

It is known that water molecules play an important role in the biological functioning of proteins. The members of the ribonuclease A (RNase A) family of proteins, which are sequentially and structurally similar, are known to carry out the obligatory function of cleaving RNA and individually perform other diverse biological functions. Our focus is on elucidating whether the sequence and structural similarity lead to common hydration patterns, what the common hydration sites are and what the differences are. Extensive molecular dynamics simulations followed by a detailed analysis of protein-water interactions have been carried out on two members of the ribonuclease A superfamily-RNase A and angiogenin. The water residence times are analyzed and their relationship with the characteristic properties of the protein polar atoms, such as their accessible surface area and mean hydration, is studied. The capacity of the polar atoms to form hydrogen bonds with water molecules and participate in protein-water networks are investigated. The locations of such networks are identified for both proteins.     

Role of water on unfolding kinetics of helical peptides studied by molecular dynamics simulations

Molecular dynamics simulations have been carried out with four polypeptides, Ala13, Val(13), Ser13, and Ala4Gly5Ala4, in vacuo and with explicit hydration. The unfolding of the polypeptides, which are initially fully alpha-helix in conformation, has been monitored during trajectories of 0.3 ns at 350 K. A rank of Ala < Val < Ser < Gly is found in the order of increasing rate of unwinding. The unfolding of Ala13 and Val(13) is completed in hundreds of picoseconds, while that of Ser13 is about one order of magnitude faster. The helix content of the peptide containing glycine residues falls to zero within a few picoseconds. Ramachandran plots indicate quite distinct equilibrium distributions and time evolution of dihedral angles in water and in vacuum for each residue type. The unfolding of polyalanine and polyvaline helices is accelerated due to solvation. In contrast, polyserine is more stable in water compared to vacuum, because its side chains can form intramolecular hydrogen bonds with the backbone more readily in vacuum, which disrupts the helix. Distribution functions of the spatial and angular position of water molecules in the proximity of the polypeptide backbone polar groups reveal the stabilization of the coiled structures by hydration. The transition from helix to coil is characterized by the appearance of a new peak in the probability distribution at a specific location characteristic of hydrogen bond formation between water and backbone polar groups. No significant insertion of water molecules is observed at the precise onset of unwinding, while (i, i+3) hydrogen bond formation is frequently detected at the initiation of alpha-helix unwinding.     

Conformational characteristics of peptides and unanticipated results from crystal structure analyses

Preferred conformation and types of molecular folding are some of the topics that can be addressed by structure analysis using x-ray diffraction of single crystals. The conformations of small linear peptide molecules with 2-6 residues are affected by polarity of solvent, presence of water molecules, hydrogen bonding with neighboring molecules, and other packing forces. Larger peptides, both cyclic and linear, have many intramolecular hydrogen bonds, the effect of which outweighs any intermolecular attractions. Numerous polymorphs of decapeptides grown from a variety of solvents, with different cocrystallized solvents, show a constant conformation for each peptide. Large conformational changes occur, however, upon complexation with metal ions. A new form of free valinomycin grown from DMSO exhibits near three-fold symmetry with only three intramolecular hydrogen bonds. The peptide is in the form of a shallow bowl with a hydrophobic exterior. Near the bottom of the interior of the bowl are three carbonyl oxygens, spaced and directed so that they are in position to form three ligands to a K+, e.g., complexation can be completed by the three lobes containing the beta-bends closing over and encapsulating the K+ ion. In another example, free antamanide and the biologically inactive perhydro analogue, in which four phenyl groups become cyclic hexyl groups, have essentially the same folding of backbone and side chains. The conformation changes drastically upon complexation with Li+ or Na+. However, the metal ion complex of natural antamanide has a hydrophobic globlar form whereas the metal ion complex of the inactive perhydro analogue has a polar band around the middle. The structure results indicate that the antamanide molecule is in a complexed form during its biological activity. Single crystal x-ray diffraction structure analyses have identified the manner in which water molecules are essential to creating minipolar areas on apolar helices. Completely apolar peptides, such as membrane-active peptides, can acquire amphiphilic character by insertion of a water molecule into the helical backbone of Boc-Aib-Ala-Leu-Aib-Ala-Leu-Aib-Ala-Leu-Aib-OMe, for example. The C-terminal half assumes an alpha-helix conformation, whereas the N-terminal half is distorted by an insertion of a water molecule W(1) between N(Ala5) and O(Ala2), forming hydrogen bonds N(5)H...W(1) and W(1)...O(2). The distortion of the helix exposes C = O(Aib1) and C = O(Aib4) to the outside environment with the consequence of attracting additional water molecules. The leucyl side chains are on the other side of the molecule. Thus a helix with an apolar sequence can mimic an amphiphilic helix.     

Statistical and molecular dynamics studies of buried waters in globular proteins

Buried solvent molecules are common in the core of globular proteins and contribute to structural stability. Folding necessitates the burial of polar backbone atoms in the protein core, whose hydrogen-bonding capacities should be satisfied on average. Whereas the residues in alpha-helices and beta-sheets form systematic main-chain hydrogen bonds, the residues in turns, coils and loops often contain polar atoms that fail to form intramolecular hydrogen bonds. The statistical analysis of 842 high resolution protein structures shows that well-resolved, internal water molecules preferentially reside near residues without alpha-helical and beta-sheet secondary structures. These buried waters most often form primary hydrogen bonds to main-chain atoms not involved in intramolecular hydrogen bonds, providing strong evidence that hydrating main-chain atoms is a key structural role of buried water molecules. Additionally, the average B-factor of protein atoms hydrogen-bonded to waters is smaller than that of protein atoms forming intramolecular hydrogen bonds, and the average B-factor of water molecules involved in primary hydrogen bonds with main-chain atoms is smaller than the average B-factor of water molecules involved in secondary hydrogen bonds to protein atoms that form concurrent intramolecular hydrogen bonds. To study the structural coupling between internal waters and buried polar atoms in detail we simulated the dynamics of wild-type FKBP12, in which a buried water, Wat137, forms one side-chain and multiple main-chain hydrogen bonds. We mutated E60, whose side-chain hydrogen bonds with Wat137, to Q, N, S or A, to modulate the multiplicity and geometry of hydrogen bonds to the water. Mutating E60 to a residue that is unable to form a hydrogen bond with Wat137 results in reorientation of the water molecule and leads to a structural readjustment of residues that are both near and distant to the water. We predict that the E60A mutation will result in a significantly reduced affinity of FKBP12 for its ligand FK506. The propensity of internal waters to hydrogen bond to buried polar atoms suggests that ordered water molecules may constitute fundamental structural components of proteins, particularly in regions where alpha-helical or beta-sheet secondary structure is not present.     

Partial molar volumes,expansibilities, and compressibilities of oligoglycines in aqueous solutions at 18–55°C

We have determined the partial molar volumes, expansibilities, and adiabatic compressibilities of glycine, diglycine, triglycine, tetraglycine, and pentaglycine over the temperature range 18–55°C. These data were analyzed and interpreted in terms of the hydration of these short oligoglycines and their constituent groups. From our results, we have estimated the contributions of the peptide group to the partial molar volume and the partial molar adiabatic compressibility of these oligoglycines. Based on these data, we propose that each of the polar atomic groups of the peptide bond forms approximately two hydrogen bonds with adjacent water molecules. Furthermore, the temperature dependence of the partial molar volume suggests that water that solvates the polar groups of a peptide linkage behaves more like a “normal” liquid than does bulk water, which exhibits its well-known anomalous liquid properties. © 1994 John Wiley & Sons, Inc.     

Free‐energy function based on an all‐atom model for proteins

We have developed a free‐energy function based on an all‐atom model for proteins. It comprises two components, the hydration entropy (HE) and the total dehydration penalty (TDP). Upon a transition to a more compact structure, the number of accessible configurations arising from the translational displacement of water molecules in the system increases, leading to a water‐entropy gain. To fully account for this effect, the HE is calculated using a statistical‐mechanical theory applied to a molecular model for water. The TDP corresponds to the sum of the hydration energy and the protein intramolecular energy when a fully extended structure, which possesses the maximum number of hydrogen bonds with water molecules and no intramolecular hydrogen bonds, is chosen as the standard one. When a donor and an acceptor (e.g., N and O, respectively) are buried in the interior after the break of hydrogen bonds with water molecules, if they form an intramolecular hydrogen bond, no penalty is imposed. When a donor or an acceptor is buried with no intramolecular hydrogen bond formed, an energetic penalty is imposed. We examine all the donors and acceptors for backbone‐backbone, backbone‐side chain, and side chain‐side chain intramolecular hydrogen bonds and calculate the TDP. Our free‐energy function has been tested for three different decoy sets. It is better than any other physics‐based or knowledge‐based potential function in terms of the accuracy in discriminating the native fold from misfolded decoys and the achievement of high Z‐scores. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Effective interactions between chaotropic agents and proteins

Chaotropic agents are cosolutes that can disrupt the hydrogen bonding network between water molecules and reduce the stability of the native state of proteins by weakening the hydrophobic effect. In this work, we represent the chaotropic agent as a factor that reduces the amount of order in the structures formed by water molecules, both in the bulk and the hydration shells around hydrophobic amino acids. In this framework we show that low chaotrope concentrations lead to a destabilization of the native state of proteins, and that high concentrations induce complete denaturation. We also find that the reduction of the number of bulk ordered states of water molecules can give origin to an effective interaction between chaotropic molecules and proteins.     

Completely buried, non-ion-paired glutamic acid contributes favorably to the conformational stability of pyrrolidone carboxyl peptidases from hyperthermophiles

Pyrrolidone carboxyl peptidases (PCPs) from hyperthermophiles have a structurally conserved and completely buried Glu192 in the hydrophobic core; in contrast, the corresponding residue in the mesophile protein is a hydrophobic residue, Ile. Does the buried ionizable residue contribute to stabilization or destabilization of hyperthermophile PCPs? To elucidate the role of the buried glutamic acid in stabilizing PCP from hyperthermophiles, we constructed five Glu192 mutants of PCP-0SH (C142S/C188S, Cys-free double mutant of PCP) from Pyrococcus furiosus and examined their thermal and pH-induced unfolding and crystal structures and compared them with those of PCP-0SH. The stabilities of apolar (E192A/I/V) and polar (E192D/Q) mutants were less than PCP-0SH at acidic pH values. In the alkaline region, the mutant proteins, except for E192D, were more stable than PCP-0SH. The thermal stability data and theoretical calculations indicated an apparent pKa value > or = 7.3 for Glu192. Present results confirmed that the protonated Glu192 in PCP-0SH forms strong hydrogen bonds with the carbonyl oxygen and peptide nitrogen of Pro168. New intermolecular hydrogen bonds in the E --> A/D mutants were formed by a water molecule introduced into the cavity created around position 192, whereas the hydrogen bonds disappeared in the E --> I/V mutants. Structure-based empirical stability of mutant proteins was in good agreement with the experimental results. The results indicated that (1) completely buried Glu192 contributes to the stabilization of PCP-0SH because of the formation of strong intramolecular hydrogen bonds and (2) the hydrogen bonds by the nonionized and buried Glu can contribute more than the burial of hydrophobic groups to the conformational stability of proteins.     

Role of flexibility and polarity as determinants of the hydration of internal cavities and pockets in proteins

Molecular dynamics simulations of Staphylococcal nuclease and of 10 variants with internal polar or ionizable groups were performed to investigate systematically the molecular determinants of hydration of internal cavities and pockets in proteins. In contrast to apolar cavities in rigid carbon structures, such as nanotubes or buckeyballs, internal cavities in proteins that are large enough to house a few water molecules will most likely be dehydrated unless they contain a source of polarity. The water content in the protein interior can be modulated by the flexibility of protein elements that interact with water, which can impart positional disorder to water molecules, or bias the pattern of internal hydration that is stabilized. This might explain differences in the patterns of hydration observed in crystal structures obtained at cryogenic and room temperature conditions. The ability of molecular dynamics simulations to determine the most likely sites of water binding in internal pockets and cavities depends on its efficiency in sampling the hydration of internal sites and alternative protein and water conformations. This can be enhanced significantly by performing multiple molecular dynamics simulations as well as simulations started from different initial hydration states.     

Crystallographic study of hydration of an internal cavity in engineered proteins with buried polar or ionizable groups

Although internal water molecules are essential for the structure and function of many proteins, the structural and physical factors that govern internal hydration are poorly understood. We have examined the molecular determinants of internal hydration systematically, by solving the crystal structures of variants of staphylococcal nuclease with Gln-66, Asn-66, and Tyr-66 at cryo (100 K) and room (298 K) temperatures, and comparing them with existing cryo and room temperature structures of variants with Glu-66, Asp-66, Lys-66, Glu-92 or Lys-92 obtained under conditions of pH where the internal ionizable groups are in the neutral state. At cryogenic temperatures the polar moieties of all these internal side chains are hydrated except in the cases of Lys-66 and Lys-92. At room temperature the internal water molecules were observed only in variants with Glu-66 and Tyr-66; water molecules in the other variants are probably present but they are disordered and therefore undetectable crystallographically. Each internal water molecule establishes between 3 and 5 hydrogen bonds with the protein or with other internal water molecules. The strength of interactions between internal polar side chains and water molecules seems to decrease from carboxylic acids to amides to amines. Low temperature, low cavity volume, and the presence of oxygen atoms in the cavity increase the positional stability of internal water molecules. This set of structures and the physical insight they contribute into internal hydration will be useful for the development and benchmarking of computational methods for artificial hydration of pockets, cavities, and active sites in proteins.     

Mechanical Unfolding of Spectrin Repeats Induces Water-Molecule Ordering

Mechanical processes are involved at many stages of the development of living cells, and often external forces applied to a biomolecule result in its unfolding. Although our knowledge of the unfolding mechanisms and the magnitude of the forces involved has evolved, the role that water molecules play in the mechanical unfolding of biomolecules has not yet been fully elucidated. To this end, we investigated with steered molecular dynamics simulations the mechanical unfolding of dystrophin’s spectrin repeat 1 and related the changes in the protein’s structure to the ordering of the surrounding water molecules. Our results indicate that upon mechanically induced unfolding of the protein, the solvent molecules become more ordered and increase their average number of hydrogen bonds. In addition, the unfolded structures originating from mechanical pulling expose an increasing amount of the hydrophobic residues to the solvent molecules, and the uncoiled regions adapt a convex surface with a small radius of curvature. As a result, the solvent molecules reorganize around the protein’s small protrusions in structurally ordered waters that are characteristic of the so-called “small-molecule regime,” which allows water to maintain a high hydrogen bond count at the expense of an increased structural order. We also determined that the response of water to structural changes in the protein is localized to the specific regions of the protein that undergo unfolding. These results indicate that water plays an important role in the mechanically induced unfolding of biomolecules. Our findings may prove relevant to the ever-growing interest in understanding macromolecular crowding in living cells and their effects on protein folding, and suggest that the hydration layer may be exploited as a means for short-range allosteric communication.